Breast milk provides not only essential nutrients for babies, but also various health benefits that are beyond the mere concept of providing nutrients. Breast milk oligosaccharides are composed of functional components, and have an oligosaccharide content of 5-10 g/liter, which is 100-200 times higher than that of cow's milk. To date, more than 130 kinds of breast milk oligosaccharides were found. The content and structural diversity of these oligosaccharides are very specific in breast milk, unlike the case of cow's milk. Among breast milk oligosaccharides, fucosyloligosaccharides are contained in cow's milk in an amount of less than 1%, but are present in breast milk in a large amount of about 50-80%. Particularly, 3-fucosyllactose, which has lactose at the reducing end and contains fucose linked to glucose by an α-1,3 bond, is not present in cow's milk, but is contained in breast milk in an amount of about 1.0 g/L.
The functions of breast milk fucosyloligosaccharides in the human body are as follows. First, breast milk fucosyloligosaccharides function as prebiotics to promote the growth of useful intestinal microorganisms such as Lactobacillus and Bifidobacteria while inhibiting the growth of harmful pathogenic microorganisms such as Clostridium. These functions are attributable to Bifidobacteria that can use short-chain fucosyloligosaccharides as a carbon source. It has been reported that these breast milk fucosyloligosaccharides can maintain the balance of such useful microbial communities, and thus these are effective for the health promotion effects such as prevention or treatment of infectious diseases, exhibition of anticancer activity, stimulation of host immune functions, and show increased vitamin intake.
Second, fucosyloligosaccharides have been studied as inhibitors that inhibit the adhesion of harmful bacteria or viruses to the intestinal epithelial surface of host cells at the initial stage of infection. 3′-Fucosyllactrose (Galβ1,4Glc(α-1,3)Fuc) can recognize receptors for Pseudomonas aeruginosa which is respiratory tract pathogen, Enterotoxigenic E. coli, Clostridium, Salmonella fyris. Thus, these fucosyloligosaccharides can competitively inhibit the invasion of these microorganisms. Breast milk fucosyloligosaccharides, including 3′-fucosyllactrose having the above-described function, can be used in various industrial applications, including baby foods, functional foods and medical drugs, etc.
In addition to various breast milk fucosyloligosaccharides, fucose is expressed in the form of sialyl-lewis X (Neu5Ac(α-2,3)Galβ1,4GlcNAc(α-1,3)Fuc) on the surface of leukocytes, and binds to lectin expressed on the surface of epithelial cells, thereby recruiting leukocytes in the initial stage of inflammation. Sialyl-Lewis X having clinical significance in immune response regulation can be used for the treatment of immune-related diseases. For example, in the case in which immune diseases in which the human immune system abnormally induces inflammatory reactions to damage autogenous tissue, sialyl-Lewis X functions as an inhibitor against the binding between leukocytes and lectin, and thus can be used as an anti-inflammatory agent.
If break milk oligosaccharides are extracted from colostrum, there is a disadvantage in that break milk oligosaccharides are difficult to produce in large amounts. In the case of the chemical synthesis of fucosyloligosaccharides, there are problems in that complex protection-deprotection reactions should be performed in order to control the three-dimensional structure of the oligosaccharides and maintain selectivity, and in that toxic reagents should be used. Due to such problems, there is a shortcoming in that it is difficult to be developed into the generalized process used in the food or pharmaceutical industry.
In such terms, synthetic technology based on bioengineering processes has been recognized as the best alternative for the economic production of oligosaccharides, and thus studies thereon have been conducted. Bioengineering processes for the production of fucosyloligosaccharides require the very expensive substrate guanosine 5′-diphosphate fucose (GDP-fuc) as a donor of fucose, and for this reason, studies on the production of guanosine 5′-diphosphate-fucose from guanosine 5′-diphosphate-mannose (GDP-mannose) or fucose and the production of fucosyloligosaccharides using fucosyltransferase have been conducted. It appears that the catalytic ability of fucosyltransferase is very important in order to efficiently transfer fucose from the fucose donor guanosine 5′-diphosphate-fucose to the receptor substrate.
α-1,3 fucosyltransferase that is used in the present invention is an enzyme that transfers fucose to carbon 3-position of glucose or N-acetylglucosamine by an α-1,3 bond, and originates from Helicobacter pylori 26695. Bernard Priem et al. removed a cytosine from polyC in accordance with the frame so that active 3-fucosyltransferase would be expressed. In addition, it was found that α-1,3 fucosyltransferase has specificity for substrates, including lactose, N-acetyllactosamine and various oligosaccharides [Claire Dumon, Assessment of the two Helicobacter pylori α-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli, Biotechnology Progress, 2004, 20, pp. 412-9].
It was reported that the C-terminus of α-1,3 fucosyltransferase from Helicobacter pylori 26695 has leucine-zipper-like 7-amino-acid repeats, and the substrate specificity of the enzyme varies depending on the number of the repeats.
Meanwhile, for the synthesis of fucosyloligosaccharides, in vivo and in vitro reactions based on both the synthesis of the fucose donor guanosine 5′-diphosphate-fucose and the reaction of fucosyltransferase have been carried out. The fucose donor guanosine 5′-diphosphate-fucose can be produced from L-fucose by the salvage pathway FKP (L-fucokinase/GDP-fucose pyrophosphorylase) enzyme having two enzymatic activities, and fucose can be transferred from the fucose donor to a receptor (e.g., lactose). In the in vivo and in vitro reactions that use the α-1,3 fucosyltransferase, the soluble protein expression level and enzymatic activity of the FKP enzyme in E. coli is higher than those of α-1,3 fucosyltransferase. Thus, in these reactions, the fucosyltransferase reaction itself was found to be a rate determining step. To enhance the fucosyltransferase reaction that is a rate determining step, it is highly required to increase the soluble protein expression and enzymatic activity of α-1,3 fucosyltransferase in E. coli. 
Until now, the total protein expression and soluble protein expression levels of Helicobacter pylori α-1,3 fucosyltransferase in E. coli have been low, and thus there has been difficulty in that the fucosyltransferase reactions in vivo and in vitro for the synthesis of fucosyloligosaccharides are slow.
α-1,3 fucosyltransferase that is used in the present invention originates from Helicobacter pylori 26695, and the C-terminus thereof has two D(D/N)LR(V/I)NY that are leucine-zipper-like 7-amino-acid repeats (heptad repeats). Helicobacter pylori α-1,3 fucosyltransferases have 2-10 heptad repeats and α-helix structures at the C-terminus, and all have problems in that the expression level of soluble protein in E. coli is very low. For this reason, with respect to α-1,3 fucosyltransferases having 8-10 heptad repeats there has been an attempt to introduce a fusion protein, or to use an expression strain capable of providing rare codon tRNA, or to truncate a portion capable of binding to the cell membrane. However, the expression levels of these α-1,3 fucosyltransferases in E. coli still remain at low levels (4-15 mg per L of culture).
The present inventors have attempted to maximize the E. coli expression level of soluble protein of the Helicobactor pylori 26695 α-1,3 fucosyltransferase having specificity not only for an N-acetylgalactosamine substrate, but also for a lactose substrate.
In order to economically produce large amounts of α-1,3 fucosyloligosaccharide by in vivo or in vitro reactions, it is required to increase enzymatic activity and the expression level of soluble protein in terms of increasing the yield and productivity of the oligosaccharides. 1,3-fucosyltransferase from Helicobactor pylori NCTC11639 is the only 1,3-fucosyltransferase whose crystal structure has been determined until now. However, until now, it has been difficult to apply an efficient screening method, due to low soluble protein expression levels. In addition, the mutagenesis on the enzyme, which employs protein engineering mutagenesis has not been attempted. Thus, the present inventors have attempted to identify a mutant having increased enzymatic activity for various receptor substrates, including lactose, compared to a wild-type strain, by use of an α-1,3 fucosyltransferase having an increased expression level of soluble protein.
It is the gist of the present invention to construct an α-1,3 fucosyltransferase mutant, which has an increased amount of soluble protein and increased activity thereof as a result of a change in the terminal structure of the protein, nucleotide sequence substitution and protein engineering mutagenesis for the production of α-1,3 fucosyloligosaccharide, and to increase the yield and productivity of α-1,3 fucosyloligosaccharides by applying the α-1,3 fucosyltransferase mutant for the production of the α-1,3 fucosyloligosaccharides while optimizing enzymatic reactions.